Fig. 4. Ratiometric pulsed alkylation-mass spectrometry analysis of Spn C101A CopY in the apo-state (A and E), the Zn₁ (per dimer) state (B and F) and the Cu₂ (per dimer) (C and G) allosteric states. rPA-MS time course profiles for AspN-derived peptide 120-131 (panels A–C) and LysC-derived peptide 83-95 containing C86 (panels E–G) (see Table 2 for exact masses). (D) Relative quantification of d₅-NEM alkylation events at C128 vs. C130 in the Zn₁ state as a function of d₅-NEM pulse time in the doubly alkylated d₅/H₅ peptide 120-131 quantified as described in Fig. S3.† Note that C130 is more reactive than C128 at all pulse times.